Crossveinless-2 Research Articles

Integrated In Silico-In Vitro Identification and Optimization of Bone Morphogenic Protein-2 Armpit Epitope as Its Antagonist Binding Site.

Bone morphogenic protein-2 (BMP-2) is the most documented member of BMP family and plays a crucial role in bone formation and growth. In this study, we systematically analyze and compare the complex crystal structures and interaction properties of BMP-2 with its cognate receptors BMPR-I/BMPR-II and with its natural antagonist crossveinless-2 (CV-2) using an integrated in silico-in vitro strategy. It is found that the antagonist-binding site is not fully overlapped with the two receptor-binding sites on BMP-2 surface; the antagonist can competitively disrupt BMP-2-BMPR-II interaction using a blocking-out-of-site manner, but has no substantial influence on BMP-2-BMPR-I interaction. Here, the antagonist-binding site is assigned as a new functional epitope armpit to differ from the traditional conformational epitope wrist and linear epitope knuckle at receptor-binding sites. Structural analysis reveals that the armpit comprises three sequentially discontinuous, structurally vicinal peptide segments, separately corresponding to a loop region and two β-strands crawling on the protein surface. The three segments cannot work independently when splitting from the protein context, but can restore binding capability to CV-2 if they are connected to a single peptide. A systematic combination of different-length polyglycine linkers between these segments obtains a series of designed single peptides, from which several peptides that can potently interact with the armpit-recognition site of CV-2 with high affinity and specificity are identified using energetic analysis and fluorescence assay; they are expected to target BMP-2-CV-2 interaction in a self-inhibitory manner.

Shaping Waves of Bone Morphogenetic Protein Inhibition During Vascular Growth.

The BMPs (bone morphogenetic proteins) are essential morphogens in angiogenesis and vascular development. Disruption of BMP signaling can trigger cardiovascular diseases, such as arteriovenous malformations. A computational model predicted that BMP4 and BMP9 and their inhibitors MGP (matrix gamma-carboxyglutamic acid [Gla] protein) and CV2 (crossveinless-2) would form a regulatory system consisting of negative feedback loops with time delays and that BMP9 would trigger oscillatory expression of the 2 inhibitors. The goal was to investigate this regulatory system in endothelial differentiation and vascular growth. Oscillations in the expression of MGP and CV2 were detected in endothelial cells in vitro, using quantitative real-time polymerase chain reaction and immunoblotting. These organized temporally downstream BMP-related activities, including expression of stalk-cell markers and cell proliferation, consistent with an integral role of BMP9 in vessel maturation. In vivo, the inhibitors were located in distinct zones in relation to the front of the expanding retinal network, as determined by immunofluorescence. Time-dependent changes of the CV2 location in the retina and the existence of an endothelial population with signs of oscillatory MGP expression in developing vasculature supported the in vitro findings. Loss of MGP or its BMP4-binding capacity disrupted the retinal vasculature, resulting in poorly formed networks, especially in the venous drainage areas, and arteriovenous malformations as determined by increased cell coverage and functional testing. Our results suggest a previously unknown mechanism of temporal orchestration of BMP4 and BMP9 activities that utilize the tandem actions of the extracellular antagonists MGP and CV2. Disruption of this mechanism may contribute to vascular malformations and disease.

Thrombospondin-1 (TSP-1), a new bone morphogenetic protein-2 and -4 (BMP-2/4) antagonist identified in pituitary cells

Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation, suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). Surface plasmon resonance and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C domain of TSP-1 is likely responsible for this BMP-2/4-binding activity, an assertion based on sequence similarity that TSP-1 shares with the von Willebrand type C domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented a structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action.

Abstract 236: Shaping Waves of BMP Inhibition During Vasculogenesis

During early embryogenesis, gradients of bone morphogenetic proteins (BMPs) have been shown to be critical in controlling patterning and growth of developing tissues. Two members of the BMP family are well known as strong modulators of vascular development, BMP4 and BMP9. Hereditary hemorrhagic telangiectasia 2 (HHT2) is caused by mutations in a BMP receptor, the activin receptor-like kinase 1 (Alk1) gene, and characterized by abnormal vascular networks and arteriovenous malformations (AVMs). ALK1 expression and signaling are tightly regulated by BMP4 and 9 and their respective inhibitors, matrix Gla protein (MGP) and Crossveinless-2 (CV2), through negative feedback regulation. Aberrant ALK1 signaling disrupting endothelial cell (EC) differentiation is closely related to vascular morphogenesis and AVMs, and depends on normal action of MGP and CV2. We have previously shown that deletion of the Mgp gene leads to AVMs in multiple organs (100% penetrance), characterized by large-caliber shunts and abnormal endothelium with increased cellularity and EC marker expression. Using in vitro , in silico and in vivo approaches, we found that the vascular growth, patterning and EC maturation are orchestrated by “shaping waves” resembling those in somatogenesis. In cultures ECs, the waves are seen as temporal oscillations of MGP and CV2 expression in response to BMP9, whereas in tissue such as the retina, the waves are observed as stripes perpendicular to the direction of growth. Thus, lack of MGP or disruptions in BMP9/ALK1/Endoglin signaling, as in HHT, will cause alterations in the expression of the BMP inhibitors and the patterning during vascular development.

Structural analyses of von Willebrand factor C domains of collagen 2A and CCN3 reveal an alternative mode of binding to bone morphogenetic protein-2

Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signaling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. Although the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC/BMP-binding mechanism, other vWC domains may bind to BMP differently. Here, using X-ray crystallography, we present for the first time structures of the vWC domains of two proteins thought to interact with BMP-2: collagen IIA and matricellular protein CCN3. We found that these two vWC domains share a similar N-terminal fold that differs greatly from that in CV-2 vWC, which comprises its BMP-2-binding site. We analyzed the ability of these vWC domains to directly bind to BMP-2 and detected an interaction only between the collagen IIa vWC and BMP-2. Guided by the collagen IIa vWC domain crystal structure and conservation of surface residues among orthologous domains, we mapped the BMP-binding epitope on the subdomain 1 of the vWC domain. This binding site is different from that previously observed in the complex between CV-2 vWC and BMP-2, revealing an alternative mode of interaction between vWC domains and BMPs.

Cyclooxygenase-2 promotes pulmonary intravascular macrophage accumulation by exacerbating BMP signaling in rat experimental hepatopulmonary syndrome

Background and aimsOne central factor in hepatopulmonary syndrome (HPS) pathogenesis is intravascular accumulation of activated macrophages in small pulmonary arteries. However, molecular mechanism underlying the macrophage accumulation in HPS is unknown. In this study, we aimed to explore whether elevated COX-2 induces the Bone morphogenic protein-2 (BMP-2)/Crossveinless-2 (CV-2) imbalance and then activation of BMP signaling pathway promotes the macrophage accumulation in Common Bile Duct Ligation (CBDL) rat lung. MethodsThe COX-2/PGE2 signaling activation, the BMP-2/CV-2 imbalance and the activation of Smad1 were evaluated in CBDL rat lung and in cultured pulmonary microvascular endothelial cells (PMVECs) under the HPS serum stimulation. The effects of Parecoxib (COX-2 inhibitor), BMP-2 and CV-2 recombinant proteins on 4-week CBDL rat lung were determined, respectively. ResultsThe COX-2/PGE2 signaling pathway was activated in CBDL rat lung in vivo and PMVECs in vitro, which was due to the activation of NF-κB P65. The inhibition of COX-2 by Parecoxib reduced macrophage accumulation, decreased lung angiogenesis and improved HPS. Meanwhile, the CBDL rat lung secreted more BMP-2 but less CV-2, and the imbalance between BMP-2 and CV-2 exacerbated the BMP signaling activation thus promoting the macrophage accumulation and lung angiogenesis. The BMP-2/CV-2 imbalance is dependent on the COX-2/PGE2 signaling pathway, and thus the effects of this imbalance can be reversed by adminstration of Parecoxib. ConclusionOur findings indicate that inhibition of COX-2 by parecoxib can improve the HPS through the repression of BMP signaling and macrophage accumulation.

Crossveinless 2 regulates bone morphogenetic protein 9 in human and mouse vascular endothelium

AbstractThe importance of morphogenetic proteins (BMPs) and their antagonists in vascular development is increasingly being recognized. BMP-4 is essential for angiogenesis and is antagonized by matrix Gla protein (MGP) and crossveinless 2 (CV2), both induced by the activin receptor like-kinase 1 (ALK1) when stimulated by BMP-9. In this study, however, we show that CV2 preferentially binds and inhibits BMP-9 thereby providing strong feedback inhibition for BMP-9/ALK1 signaling rather than for BMP-4/ALK2 signaling. CV2 disrupts complex formation involving ALK2, ALK1, BMP-4, and BMP-9 required for the induction of both BMP antagonists. It also limits VEGF expression, proliferation, and tube formation in ALK1-expressing endothelial cells. In vivo, CV2 deficiency translates into a dysregulation of vascular BMP signaling, resulting in an abnormal endothelium with increased endothelial cellularity and expression of lineage markers for mature endothelial cells. Thus, mutual regulation by BMP-9 and CV2 is essential in regulating the development of the vascular endothelium.

Abstract 210: Crossveinless 2 Regulates Bone Morphogenetic Protein 9 in Human and Mouse Vascular Endothelium

The importance of morphogenetic proteins (BMPs) and their antagonists in vascular development is increasingly being recognized. BMP-4 is essential for angiogenesis and is antagonized by matrix Gla protein (MGP) and crossveinless 2 (CV2), both induced in a staged fashion by the activin-like kinase receptor 1 (ALK1) after stimulation by BMP-9. In this study, however, we show that CV2 preferentially binds and inhibits BMP-9 thereby providing strong feedback inhibition for BMP-9/ALK1 signaling rather than for BMP-4/ALK2 signaling. CV2 disrupts complex formation by ALK2, ALK1, BMP-4 and BMP-9 required for the induction of both BMP antagonists. It also limits VEGF expression and proliferation of ALK1-expressing endothelial cells. In vivo, CV2 deficiency translates into a dysregulation of vascular BMP signaling, resulting in a thickened, abnormal endothelium with increased markers of endothelial differentiation. Thus, mutual regulation by BMP-9 and CV2 is essential in regulating the development of the vascular endothelium.

Binding between Crossveinless-2 and Chordin Von Willebrand Factor Type C Domains Promotes BMP Signaling by Blocking Chordin Activity

BackgroundCrossveinless-2 (CV2) is an extracellular BMP modulator protein of the Chordin family, which can either enhance or inhibit BMP activity. CV2 binds to BMP2 via subdomain 1 of the first of its five N-terminal von Willebrand factor type C domains (VWC1). Previous studies showed that this BMP binding is required for the anti-, but not for the pro-BMP effect of CV2. More recently, it was shown that CV2 can also bind to the BMP inhibitor Chordin. However, it remained unclear which domains mediate this binding, and whether it accounts for an anti- or pro-BMP effect.Principal FindingsHere we report that a composite interface of CV2 consisting of subdomain 2 of VWC1 and of VWC2-4, which are dispensable for BMP binding, binds to the VWC2 domain of Chordin. Functional data obtained in zebrafish embryos indicate that this binding of Chordin is required for CV2's pro-BMP effect, which actually is an anti-Chordin effect and, at least to a large extent, independent of Tolloid-mediated Chordin degradation. We further demonstrate that CV2 mutant versions that per se are incapable of BMP binding can attenuate the Chordin/BMP interaction.ConclusionsWe have physically dissected the anti- and pro-BMP effects of CV2. Its anti-BMP effect is obtained by binding to BMP via subdomain1 of the VWC1 domain, a binding that occurs in competition with Chordin. In contrast, its pro-BMP effect is achieved by direct binding to Chordin via subdomain 2 of VWC1 and VWC2-4. This binding seems to induce conformational changes within the Chordin protein that weaken Chordin's affinity to BMP. We propose that in ternary Chordin-CV2-BMP complexes, both BMP and Chordin are directly associated with CV2, whereas Chordin is pushed away from BMP, ensuring that BMPs can be more easily delivered to their receptors.

Crossveinless-2 is required for the relocalization of Chordin protein within the vertebral field in mouse embryos

Bone morphogenetic proteins (BMPs), as well as the BMP-binding molecules Chordin (Chd), Crossveinless-2 (CV2) and Twisted Gastrulation (Tsg), are essential for axial skeletal development in the mouse embryo. We previously reported a strong genetic interaction between CV2 and Tsg and proposed a role for this interaction in the shaping of the BMP morphogenetic field during vertebral development. In the present study we investigated the roles of CV2 and Chd in the formation of the vertebral morphogenetic field. We performed immunostainings for CV2 and Chd protein on wild-type, CV2−/− or Chd−/− mouse embryo sections at the stage of onset of the vertebral phenotypes. By comparing mRNA and protein localizations we found that CV2 does not diffuse away from its place of synthesis, the vertebral body. The most interesting finding of this study was that Chd synthesized in the intervertebral disc accumulates in the vertebral body. This relocalization does not take place in CV2−/− mutants. Instead, Chd was found to accumulate at its site of synthesis in CV2−/− embryos. These results indicate a CV2-dependent flow of Chd protein from the intervertebral disc to the vertebral body. Smad1/5/8 phosphorylation was decreased in CV2−/−vertebral bodies. This impaired BMP signaling may result from the decreased levels of Chd/BMP complexes diffusing from the intervertebral region. The data indicate a role for CV2 and Chd in the establishment of the vertebral morphogenetic field through the long-range relocalization of Chd/BMP complexes. The results may have general implications for the formation of embryonic organ-forming morphogenetic fields.

Extracellular regulation of BMP signaling

In the developing organism, cells differentiate, divide and die as part of groups of hundreds or thousands of cells called 'morphogenetic fields'. Fields have the remarkable property of self-regulation: for example, if the forelimb field is bisected, each half can give rise to a complete limb after transplantation, as discovered by Ross Harrison in 1918. Therefore, cells in the morphogenetic field are capable of long-range communication with each other in order to ascertain their position [1]. This positional information is relayed in the extracellular space in the form of concentration gradients of specific classes of extracellular molecules called 'morphogens' that trigger cellular responses by binding and activating cell surface receptors. Here, we focus on a family of morphogens called 'Bone Morphogenetic Proteins' (BMPs), which has provided a new paradigm for signaling regulation in the extracellular space.

Systems biology of the self-regulating morphogenetic gradient of the Xenopus gastrula.

The morphogenetic field concept was proposed by experimental embryologists to account for the self-regulative behavior of embryos. Such fields have remained an abstract concept until the recent identification of their molecular components using a combination of genetics, biochemistry, and theoretical modeling. One of the best studied models of a morphogenetic field is the Dorsal-Ventral (D-V) patterning of the early frog embryo. This patterning system is regulated by the bone morphogenetic protein (BMP) signaling pathway and an intricate network of secreted protein antagonists. This biochemical pathway of interacting proteins functions in the extracellular space to generate a D-V gradient of BMP signaling, which is maintained during extensive morphogenetic movements of cell layers during gastrulation. The D-V field is divided into a dorsal and a ventral center, in regions of low and high BMP signaling respectively, under opposite transcriptional control by BMPs. The robustness of the patterning is assured at two different levels. First, in the extracellular space by secreted BMP antagonists that generate a directional flow of BMP ligands to the ventral side. The flow is driven by the regulated proteolysis of the Chordin inhibitor and by the presence of a molecular sink on the ventral side that concentrates BMP signals. The tolloid metalloproteinases and the Chordin-binding protein Crossveinless-2 (CV2) are key components of this ventral sink. Second, by transcriptional feedback at the cellular level: The dorsal and ventral signaling centers adjust their size and level of BMP signaling by transcriptional feedback. This allows cells on one side of a gastrula containing about 10,000 cells to communicate with cells in the opposite pole of the embryo.

Crossveinless-2 Is a BMP Feedback Inhibitor that Binds Chordin/BMP to Regulate Xenopus Embryonic Patterning

Vertebrate Crossveinless-2 (CV2) is a secreted protein that can potentiate or antagonize BMP signaling. Through embryological and biochemical experiments we find that: (1) CV2 functions as a BMP4 feedback inhibitor in ventral regions of the Xenopus embryo; (2) CV2 complexes with Twisted gastrulation and BMP4; (3) CV2 is not a substrate for tolloid proteinases; (4) CV2 binds to purified Chordin protein with high affinity (K(D) in the 1 nM range); (5) CV2 binds even more strongly to Chordin proteolytic fragments resulting from Tolloid digestion or to full-length Chordin/BMP complexes; (6) CV2 depletion causes the Xenopus embryo to become hypersensitive to the anti-BMP effects of Chordin overexpression or tolloid inhibition. We propose that the CV2/Chordin interaction may help coordinate BMP diffusion to the ventral side of the embryo, ensuring that BMPs liberated from Chordin inhibition by tolloid proteolysis cause peak signaling levels.

Crystal Structure Analysis Reveals How the Chordin Family Member Crossveinless 2 Blocks BMP-2 Receptor Binding

Crossveinless 2 (CV-2) is an extracellular BMP modulator protein belonging to the Chordin family. During development it is expressed at sites of high BMP signaling and like Chordin CV-2 can either enhance or inhibit BMP activity. CV-2 binds to BMP-2 via its N-terminal Von Willebrand factor type C (VWC) domain 1. Here we report the structure of the complex between CV-2 VWC1 and BMP-2. The tripartite VWC1 binds BMP-2 only through a short N-terminal segment, called clip, and subdomain (SD) 1. Mutational analysis establishes that the clip segment and SD1 together create high-affinity BMP-2 binding. All four receptor-binding sites of BMP-2 are blocked in the complex, demonstrating that VWC1 acts as competitive inhibitor for all receptor types. In vivo experiments reveal that the BMP-enhancing (pro-BMP) activity of CV-2 is independent of BMP-2 binding by VWC1, showing that pro- and anti-BMP activities are structurally separated in CV-2.

Crystallization and preliminary X-ray analysis of the complex of the first von Willebrand type C domain bound to bone morphogenetic protein 2

Crossveinless 2 (CV2) is a member of the chordin family, a protein superfamily that modulates the activity of bone morphogenetic proteins such as BMP2. The BMPs represent a large group of secreted proteins that control many steps during embryonal development and in tissue and organ homeostasis in the adult organism. The gene encoding the first von Willebrand type C domain (VWC1) of CV2 was cloned, expressed in Escherichia coli and purified to homogeneity. The binary complex of CV2 VWC1 and BMP2 was purified and subjected to crystallization. Crystals of SeMet-labelled proteins were obtained in two different forms belonging to the tetragonal space groups P4(1)2(1)2 and I4(1), with unit-cell parameters a = b = 86.7, c = 139.2 A and a = b = 83.7, c = 139.6 A, respectively. Initial analysis suggests that a complete binary complex consisting of one BMP2 dimer bound to two CV2 VWC1 domains is present in the asymmetric unit.

Von Willebrand Factor Type C Domain-containing Proteins Regulate Bone Morphogenetic Protein Signaling through Different Recognition Mechanisms

Bone morphogenetic protein (BMP) function is regulated in the extracellular space by many modulator proteins, including those containing a von Willebrand factor type C (VWC) domain. The function of the VWC domain-containing proteins in development and diseases has been extensively studied. The structural basis, however, for the mechanism by which BMP is regulated by these proteins is still poorly understood. By analyzing chordin, CHL2 (chordin-like 2), and CV2 (crossveinless 2) as well as their individual VWC domains, we show that the VWC domain is a versatile binding module that in its multiple forms and environments can expose a variety of binding specificities. Three of four, two of three, and one of five VWCs from chordin, CHL2, and CV2, respectively, can bind BMPs. Using an array of BMP-2 mutant proteins, it can be demonstrated that the binding-competent VWC domains all use a specific subset of BMP-2 binding determinants that overlap with the binding site for the type II receptors (knuckle epitope) or for the type I receptors (wrist epitope). This explains the competition between modulator proteins and receptors for BMP binding and therefore the inhibition of BMP signaling. A subset of VWC domains from CHL2 binds to the Tsg (twisted gastrulation) protein similar to chordin. A stable ternary complex consisting of BMP-2, CHL2, and Tsg can be formed, thus making CHL2 a more efficient BMP-2 inhibitor. The VWCs of CV2, however, do not interact with Tsg. The present results show that chordin, CHL2, and CV2 regulate BMP-2 signaling by different recognition mechanisms.